New Results

Microtubule-associated IQD9 guides cellulose synthase velocity to shape seed mucilage

Bo Yang, Gina Stamm, Katharina Bürstenbinder, Cătălin Voiniciuc
doi: https://doi.org/10.1101/2021.12.11.472226

Summary

  • Arabidopsis seeds release large capsules of mucilaginous polysaccharides, which are shaped by an intricate network of cellulosic microfibrils. Cellulose synthase complexes is guided by the microtubule cytoskeleton, but it is unclear which proteins mediate this process in the seed coat epidermis (SCE).

  • Using reverse genetics, we identified IQ67 DOMAIN 9 (IQD9) and KINESIN LIGHT CHAIN-RELATED 1 (KLCR1) as two highly expressed genes during seed development and comprehensively characterized their roles for cell wall polysaccharide biosynthesis and cortical microtubule (MT) organization.

  • Mutations in IQD9 as well as in KLCR1 lead to compact mucilage capsules with aberrant cellulose distribution, which can be rescued by transgene complementation. Double mutant analyses revealed that their closest paralogs (IQD10 and KLCR2, respectively) are not required for mucilage biosynthesis. IQD9 physically interacts with KLCR1 and localizes to cortical MTs to maintain their organization in SCE cells. Similar to the previously identified TONNEAU1 (TON1) RECRUITING MOTIF 4 (TRM4) protein, IQD9 is required to maintain the velocity of cellulose synthases.

  • Our results demonstrate that IQD9, KLCR1 and TRM4 are MT-associated proteins that are required for seed mucilage architecture. This study provides the first direct evidence that members of the IQD, KLCR and TRM families have overlapping roles in guiding the distribution of cell wall polysaccharides. Therefore, SCE cells provide an attractive system to further decipher the complex genetic regulation of polarized cellulose deposition.

Introduction

The seed coat epidermal (SCE) cells of some Angiosperms, including the model plant Arabidopsis thaliana, synthesize large amounts of hydrophilic polysaccharides (North et al., 2014; Voiniciuc et al., 2015c; Šola et al., 2019). Although the mucilage capsules that rapidly encapsule Arabidopsis seeds upon hydration are pectin-rich, they can be regarded as specialized secondary cell walls because they also contain hemicelluloses that are typical of woody tissues (Voiniciuc et al., 2015c). Substituted xylans and heteromannans maintain the attachment of mucilaginous pectin to the seed surface and the organization of ray-like cellulose microfibrils (Yu et al., 2014; Voiniciuc et al., 2015b,a; Hu et al., 2016; Ralet et al., 2016). Upon imbibition of a dry seed, expanding mucilage ruptures the outer primary cell wall to release a two-layered gelatinous capsule that can be visualized by ruthenium red (RR), a pectin-binding dye. Cellulosic rays extend from the top of each SCE cell to intertwine and anchor the inner, adherent mucilage layer to the seed surface (Sullivan et al., 2011; Mendu et al., 2011; Harpaz-Saad et al., 2011). However, the genetic factors that modulate the deposition of highly ordered cellulosic structures in seed mucilage remain largely unknown.

The current dogma is that plant crystalline microfibrils are produced by rosette-shaped cellulose synthase (CESA) complexes (CSC) composed of at least three different CESA isoforms and a growing number of interacting proteins (Polko & Kieber, 2019). In Arabidopsis SCE cells, mutations in CESA3 and CESA5 have been shown to affect the deposition of cellulose in mucilage pockets. Loss-of-function cesa5 mutants have a nearly complete loss of adherent mucilage due to reduced cellulose production (Sullivan et al., 2011; Mendu et al., 2011; Harpaz-Saad et al., 2011), while cesa3 missense mutants lead to milder alterations of mucilage adherence and cellulose organization (Griffiths et al., 2015). Several accessory proteins are also known to influence mucilage cellulose synthesis. COBRA-LIKE2 (COBL2) contains a glycosyl-phosphatidylinositol (GPI) anchor and facilitates the assembly of crystalline cellulose by CESA5 (Ben-Tov et al., 2015), while FEI2 (meaning “fat” in Chinese) and SALT-OVERLY SENSITIVE5 (SOS5) mediate pectin adherence to cellulosic rays via an independent mechanism (Griffiths et al., 2014, 2016; Ben-Tov et al., 2018). CSC assembly and trafficking are maintained by STELLOs (Greek for “to send”; Zhang et al., 2016), and negatively regulated by SHOU4 (“thin” in Chinese; Polko et al., 2018), identified via a screen for fei2 suppressors.

Cortical microtubules (MTs) can orient cellulose microfibril deposition by positioning the delivery of CSCs to the plasma membrane (PM) and guiding their subsequent trajectories (Paredez et al., 2006; Gutierrez et al., 2009; Bringmann et al., 2012a). MT-associated proteins, which shape the cytoskeleton in response to environmental or developmental signals (Lloyd & Hussey, 2001; Sedbrook & Kaloriti, 2008), can influence the organization of mucilage polysaccharides. A temperature-sensitive point mutation in MICROTUBULE ORGANIZATION 1 (MOR1) significantly reduced mucilage release at 29°C (McFarlane et al., 2008). A second MT-associated protein, TONNEAU1 (TON1) RECRUITING MOTIF 4 (TRM4), was recently found to organize cortical arrays and cellulose distribution (Yang et al., 2019). The trm4 seed mucilage capsules are compact and have shorter cellulosic rays compared to the WT, without altering pectin adherence. Arabidopsis CESAs circle around the cytoplasmic column of SCE cells to polarly deposit cellulose microfibrils in mucilage pockets (Griffiths et al., 2015), but the network of proteins that guide CESAs in SCE cells remains unclear.

Plant-specific IQ67 DOMAIN (IQD) proteins associate with MTs and have scaffold-like properties (Bürstenbinder et al., 2017). Their eponymous IQ67 domain contains 67 amino acids with calmodulin-recruiting motifs (Abel et al., 2005), which could be involved in Ca2+ signaling integration (Kölling et al., 2019). Since multiple Arabidopsis IQDs control cell shape and size (Bürstenbinder et al., 2017; Liang et al., 2018; Mitra et al., 2019), they are hypothesized to support cell wall deposition. Certain IQDs interact with KINESIN LIGHT CHAIN-RELATEDs (KLCRs; Bürstenbinder et al., 2013; Zang et al., 2021) proteins. KLCRs are also known as CELLULOSE SYNTHASE-MICROTUBULE UNCOUPLING (CMU) and stabilize MTs at the PM during cellulose synthesis (Liu et al., 2016). However, direct evidence for how IQDs influence the biosynthesis of cell wall polysaccharides has been lacking.

In this study, we identified IQD9 and KLCR1 as two additional players that maintain MT organization and the deposition of ray-like cellulosic microfibrils in SCE cells. In the absence of IQD9 or KLCR1, mutant seeds released compact mucilage capsules due to aberrant deposition of cellulose microfibrils as previously observed for trm4 lines. IQD9 physically interacted in vivo with KLCR1 and formed filamentous arrays. Live-cell imaging showed that IQD9 and KLCR1 are localized a circular pattern during mucilage biosynthesis, which is reminiscent of CSC trajectories. Both iqd9 and trm4 mutants displayed slower CESA3 particles in SCE cells, indicating that these MT-associated proteins guide cellulose deposition. IQD, KLCR and TRM proteins therefore have overlapping roles in cell wall biosynthesis.

Materials and Methods

Plant materials and growth conditions

Arabidopsis thaliana Col-0 (WT) and T-DNA insertion mutants (Table S1) analyzed in this study were obtained from the Nottingham Arabidopsis Stock Centre, unless otherwise noted. The proUBQ:RFP-TUB6 in Col-0 (Ambrose et al., 2011), proCESA3:GFP-CESA3 in je5 mutant (Desprez et al., 2007) and pKLCR1:KLCR1-GFP in klcr1-1 (Zang et al., 2021) transgenic plants have been described previously. Arabidopsis plants were grown in a phytochamber with constant light (100–120 µmol m-2 s-1), 22°C and 60% humidity. Nicotiana benthamiana plants were grown in a greenhouse (16 h light, 8 h dark) at 22–24°C.

Arabidopsis transcriptional analyses

Total RNA was isolated from 10-day-old seedlings using TRI Reagent (Sigma Aldrich), according to its manual. The cDNA was prepared from 4 µg of RNA using RevertAid reverse Transcriptase (Thermo Fisher Scientific), and reverse transcription polymerase chain reaction (RT-PCR) was performed using primers listed in Table S2. ACTIN2 served as a housekeeping gene, and WT genomic DNA was included as a control.

For GUS staining, sample tissues (seedlings, flower buds, siliques) were fixed in 80% (v/v) ice-cold acetone for 30 min and incubated for 4 h to overnight in GUS staining solution (50 mM sodium phosphate, pH 7.2, 0.5 mM K3Fe(CN)6, 0.5 mM K4Fe(CN)6, 2 mM 5-bromo-4-chloro-3-indolyl-β-glucuronic acid, 10 mM EDTA) at 37 °C. Images were acquired using a Zeiss Axioplan 2 microscope or a Nikon SMZ 1000. For GUS staining of Arabidopsis seeds, siliques of different ages were opened with forceps at the replum. Seeds were collected with a small spoon and transferred to a 1.5 ml tube with staining solution. Prior to DIC microscopy, staining solution was removed and samples were mounted in chloralhydrate.

Plasmid construction and plant transformation

Primers used for plasmid construction are listed in Table S2. Promoter:GFP-GUS constructs were generated by integrating 1.4 kb of the IQD9 or IQD10 promoter into pBGWFS7 (Karimi et al., 2002). The pIQD9:IQD9-GFP transgene was assembled using full-length genomic DNA (from ∼1.5 kb upstream of ATG up to, but excluding, the IQD9 stop codon) into pB7FWG0 vector. The constructs were stably transformed into Arabidopsis WT plants (for pIQD:GFP-GUS) or iqd9 iqd10 double mutant (for pIQD9:IQD9-GFP) respectively via Agrobacterium-mediated floral dip transformation. The p35S:IQD9-GFP, p35S:mCherry-KLCR1, and p35S:RFP-KLCR1 transgenes for N. benthamiana assays were cloned using Gateway into pB7FWG2 (Karimi et al., 2002), pJOG393 (Gantner et al., 2018) or pGWB455 (Nakagawa et al., 2007) vectors, respectively. The mCherry- and RFP-tagged KLCR1 showed similar results. The pUBQ10:RFP-TUB6 plasmid for transient expression was previously generated (Yang et al., 2019). Transient expression was performed in N. benthamiana leaves as previously described (Grefen et al., 2010). In short, Agrobacterium tumefaciens GV3101 cells containing the desired constructs were mixed with the P19 viral suppressor (OD600 = 0.7 for each) and incubated for 4 h (at 18 °C, 200 rpm) before infiltration into the lower side of leaves from 5-week-old plants.

Staining and quantification of mucilage area

Around 30 seeds were hydrated in water for 30 min and stained with 300 µl of 0.01% (w/v) RR (Sigma-Aldrich; R2751) for 15 min at 125 rpm in 24-well plates. After rinsing with water, the stained seeds were re-suspended in 300 µl of water and imaged with a Leica M165FC stereomicroscope equipped with MC170 HD camera. The mucilage and seed projected areas were quantified using an existing ImageJ pipeline (Voiniciuc et al., 2015b).

Cellulose around hydrated seeds was stained with 0.01% (w/v) Pontamine fast scarlet 4B (S4B, also known as Direct Red 23 [Sigma-Aldrich; 212490]) in 50 mM NaCl for 60 min at 125 rpm in 24-well plates (Anderson et al., 2010; Mendu et al., 2011). The counterstain was performed by mixing with 25 ug mL-1 Calcofluor (Megazyme C-CLFR) for 5 min. Seeds were imaged with a Carl Zeiss LSM 780 microscope with 10X/0.45 objective and the following excitation / emission wavelengths (S4B: 561 / 580–650 nm; Calcofluor: 405 / 410– 452 nm). The lengths of cellulosic rays were measured by ImageJ.

To view surface morphology, around 30 seeds were mixed with 500 µL of 0.01% (w/v) propidium iodide for 15 min. Seeds were rinsed twice with water and imaged using a Leica LSM 900 with 10X/0.3 objective (excitation 488 nm, emission 600-650 nm).

Seed polysaccharide quantification

Non-adherent mucilage was extracted by gently mixing 5 mg seeds in water for 30 min at 125 rpm and subsequently the adherent mucilage was isolated using a ball mill (Retsch; MM400) for 30 min at 30 Hz, as previously described (Voiniciuc, 2016). The two mucilage fractions, spiked with ribose and inositol respectively, were hydrolyzed and quantified via high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD), as described (Voiniciuc & Günl, 2016) with the following changes. HPAEC-PAD was performed on a Metrohm 940 Professional IC Vario, using Metrosep Carb 2-250/4.0 columns and a published gradient (Mielke et al., 2021). Peaks were integrated and calibrated (manually corrected if necessary) in the MagIC Net 3.2 software (Metrohm).

Crystalline cellulose was quantified using the Updegraff reagent (Updegraff, 1969) and the anthrone colorimetric assay (Foster et al., 2010), as previously adapted for Arabidopsis whole seeds (Voiniciuc et al., 2015b).

Salt stress treatments

Germination assays were performed in 24-well culture plates as described previously (Yang et al., 2021). Around 35 seeds were hydrated in 500 µl of water or 150 mM CaCl2 solution per well. All the seeds were vernalized for 66 h (dark, 4°C), transferred to a chamber with constant light (100– 120 µmol m-2 s-1), 22°C and 60% humidity. The seeds were imaged every 24 h with a Leica M165FC stereomicroscope and defined as germinated when the radicle length was > 70 µm.

For the seedling salt stress assay, the seeds were placed on ½ Murashige and Skoog agar plates, stratified for 66 h (dark, 4°C) and grown vertically in the climate-controlled chamber described above. Five-day-old seedlings of similar size were transferred to fresh agar plates with or without 100 mM NaCl and growth was imaged using a Nikon D5600 digital camera.

Stem cell wall analyses

Stem sections were cut by hand from the basal third of stems of 6-week-old plants and were stained in 0.01% toluidine blue O (Sigma-Aldrich; T3260) for 2 min. Stained sections were rinsed twice with water and imaged on a Axioplan 2 with Axiovision software (Zeiss).

The bottom 7 cm of mature stems were harvested and homogenized using a ball mill (Retsch MM400) for 10 min at 30 Hz. The AIR was extracted by sequential washes with 1 mL of 70% (v/v) ethanol, 1 mL of 1:1 (v/v) chloroform:methanol and 1 mL of acetone. Stem AIR was hydrolyzed and quantified via HPAEC-PAD exactly as described for seed mucilage, using ribose as the internal standard.

Confocal microscopy and image analysis

For fluorescence co-localization assays in N. benthamiana, leaf discs at 3 days post-infiltration were imaged with a Zeiss LSM880 inverted confocal microscope using a 40X/1.2 water-immersion objective. For oryzalin treatment, small pieces of transfected leaf were immersed with 1% (v/v) dimethylsulfoxide (DMSO) as mock treatment or 1% (v/v) DMSO containing 0.1 mM oryzalin (Sigma-Aldrich) for 4 h.

For the fluorescence imaging of the Arabidopsis SCE cells, seeds were carefully dissected from siliques at specific stages and were imaged using a Zeiss LSM880 in Airyscan mode with a 40X/1.2 water-immersion objective. For PM, 7 DPA (days post-anthesis) seeds were pre-stained in 50 µM FM4-64 for 30 min before imaging. The subcellular localization of GFP-CESA3 was detected using a 63X/1.4 oil-immersion objective and Airyscan mode, with 330 ms exposure time based on previously described protocols (Vellosillo et al., 2015; Duncombe et al., 2020). Unless stated otherwise, the time-lapse series were acquired every 5 sec for 5 min. All the samples were mounted on confocal dishes with spacers (VWR International; 734-2905) and were examined with the following excitation/emission settings: GFP (488 / 505–530 nm), RFP/mCherry (514 / 580–635 nm), FM4-64 (514 / 600–700 nm).

For hypocotyl imaging, seeds were sowed on ½ MS agar plates and stratified for 66 h (dark, 4°C). The plates were exposure to light for 6 h at room temperature and then were wrapped with aluminum foil to keep the plates in the dark at room temperature. For the observation of RFP-TUB6 in the hypocotyl epidermal cells, the inner face of epidermal cells in zone 1 of 4-d-old dark-grown seedlings were examined as described previously (Crowell et al., 2011).

All images were processed uniformly using ImageJ. The maximum projection of the Z-stack or time-lapse view was generated by frames using the Z Project tool and average intensity. For the colocalization evaluation, the intensity plot analysis was done by “RGB Profile Plot” plugin. The Pearson correlation coefficient of region of interest (ROI) from single frame was quantified with “Coloc 2”. Kymograph analysis of proteins and velocity quantification of GFP-CESA3 were performed as previously described (Vellosillo et al., 2015). Briefly, the time-lapse stack was generated with “Walking Average” plugin. The GFP-CESA3 track was depicted using segmented line on time average image and transferred to time-lapse stack. The kymographs were generated using the “MultipleKymograph” plugin, and the slope of each line was used to calculate the particle velocity.

Protein-Protein Interactions

Proteins were extracted from transiently transformed N. benthamiana leaves using 1 ml lysis buffer (20 mM HEPES, pH 7.5, 40 mM KCl, 1 mM EDTA, 0.1% [v/v] Triton X-100, 1× protease inhibitor cocktail, 1 mM phenylmethylsulfonyl fluoride, and 10% [v/v] glycerol) (Ganguly et al., 2020). Homogenate from 500 mg of plant material was centrifuged twice at 4°C at 15000 g for 10 min, and 900 µl of the supernatant was incubated with 20 µl GFP-Traps (Chromotek) overnight at 4°C on a rocking shaker. Beads were equilibrated following manufacturer’s manual using lysis buffer, without Triton-X. The next day, beads were washed four times and were boiled in 80 µl 2x Laemmli Buffer. For each sample, 40 µl were loaded on a SDS gel and blotted afterwards for 1 h. For protein detection, 1:1000 dilution of the GFP antibody 3H9 (Chromotek) or the RFP antibody 6G6 (Chromotek) was used. Anti-mouse (Sigma A9044 1:20000) and anti-rat (Thermo 31470 1:3000) secondary antibodies conjugated to horseradish peroxidase were used to detect RFP and GFP signals, respectively. Western blot images were acquired with a FluorChem system, using 1:1 mixture of Amersham ECL-Prime and ECL-Select as chemiluminescent detection reagents.

Results

IQD9 and KLCR1 control seed mucilage architecture

We hypothesized that one or more IQD genes are involved in Arabidopsis seed mucilage biosynthesis and screened their expression profiles using published transcriptional datasets. IQD9 (At2G33990) and IQD10 (At3G15050), its closest paralog, were upregulated in the seed coat at the stages of mucilage biosynthesis (Fig. 1a; Winter et al., 2007; Le et al., 2010). T-DNA insertions in these genes disrupted their transcription (Fig. 1b) and were screened for seed mucilage defects. While iqd10 mutant seeds resembled WT, two independent iqd9 mutants displayed compact RR-stained mucilage capsules (Fig. 1c). Consistent with microarray data and the seed mucilage defects (Fig. 1), promoter:GUS reporter assays only showed high seed coat activity for pIQD9 (Fig. S1). IQD9 and IQD10 displayed partially overlapping expression profiles but share only 47% amino acid identity (Fig. S1). Furthermore, iqd9-1 iqd10-1 (i9 i10) double mutant seeds showed compact mucilage capsules resembling the iqd9 single mutants (Fig. 1c).

Fig. 1
Fig. 1

Mutation of IQD9 and KLCR1 caused compact mucilage capsule. (a) Expression profiles in the seed eFP browser (Winter et al., 2007; Le et al., 2010), including absolute expression values. Glob. (globular); Cotyl. (cotyledon). (b) UTR, intron and exon structure of candidate genes. The position and the effects of T-DNA insertions were verified using RT-PCR, ACTIN2 as a reference gene, g as genomic DNA control, and – as no DNA control. Transgene complemented were marked by + IQD or + KLCR. Scale bars for gene models = 1000 bp. (c) RR staining of adherent mucilage capsules after gentle shaking in water. Bars = 100 µm. (d) Seed (triangles) and RR-stained mucilage (black dots) area of four biological replicates (>20 seeds each) per genotype. Boxes show the 25–75% quartiles, the median value (inner horizontal line), and whiskers extending to the largest/smallest values. Different letters mark P < 0.01 for one-way ANOVA with Tukey test. (e) Absolute amounts of monosaccharides in sequentially-extracted mucilage fractions. Data show mean ±SD of 5 biological replicates, and asterisks mark significant changes compared to WT (Student’s t-test, P < 0.001). See Fig. 2 for detailed composition.

Since IQD-KLCR interactions have been reported (Bürstenbinder et al., 2013), we also assessed if KLCR1/CMU1 (At4g10840) and KLCR2/CMU2 (At3g27960) are involved in mucilage biosynthesis. KLCR1 was highly expressed throughout the seed development, while KLCR2 transcription peaks at the pre-globular and globular stages, before mucilage biosynthesis (Fig. S2a). Two knockout klcr1 alleles, klcr1-1 and cmu1 (Fig. S2b, Table S1), resembled the iqd9 compact mucilage defect (Fig. 1a), while klcr2 seeds displayed WT-like mucilage. Both iqd9 as well as klcr1 mutants reduced mucilage capsule area by 30–40% compared to WT (Fig. 1d), without altering seed size. The double mutant i9 i10 and klcr1-1 klcr2-2 (k1 k2) phenocopied the mucilage structure of iqd9-1 and klcr1-1 respectively, indicating no functional redundancy between the related genes. Transgene complementation of i9 i10 with IQD9 and of klcr1-1 with KLCR1 fully rescued the compact mucilage defects (Fig. 1). Both iqd9 and klcr1 mutants resembled the mucilage phenotype of trm4, which has SCE cells with disorganized MTs (Yang et al., 2019). Moreover, iqd9 klcr1 and iqd9 trm4 double mutants displayed compact RR-stained mucilage capsules equivalent to the single mutants (Fig. S2c–f).

IQD9 and KLCR1 are specifically required for cellulose distribution in mucilage

Monosaccharide analysis of non-adherent and adherent mucilage fractions revealed that the iqd9, klcr1 and trm4 mutants did not alter the content or adherence of matrix polysaccharides to the seed surface (Fig. 1e). Surprisingly, despite no impact on RR staining, the iqd10-1 mutation correlated with a small but statistically significant reduction in total extractable monosaccharides (Fig. 1c–e). The iqd10-1 mutation decreased rhamnose (Rha) and galacturonic acid (GalA) content in non-adherent mucilage, along with galactose (Gal) and arabinose (Ara) in adherent mucilage (Fig. 2). Nevertheless, compared to the galactoglucomannan-deficient muci10 mutant with compact mucilage, all the MT-related mutant seeds released relatively normal levels of mucilage glycans (Fig. 2).

Fig. 2
Fig. 2

Composition of matrix polysaccharides in seed mucilage extracts. (a) Non-adherent and (b) adherent mucilage polysaccharides were sequentially extracted using water and different mixing intensities. Data show mean + SD of 5 biological replicates (only 3 for muci10). Red asterisks and arrows (for galactoglucomannan subunits) mark differences from WT (Student’s t-test, P < 0.0001).

Since the matrix polysaccharide composition could not account for the compact mucilage defects of iqd9 and klcr1, we then examined the structure of cellulose using S4B, a specific fluorescent dye (Anderson et al., 2010). The iqd9 and klcr1 mutant seeds extruded less cellulose upon hydration (Fig. 3a) and had ∼30% shorter rays atop each columella compared with WT (Fig. 3b). Moreover, the mutant seeds lacked the diffuse cellulose staining that was observed between the WT rays. The S4B-stained seeds of i9 i10 and k1 k2 resembled the iqd9 and klcr1 single mutants, while iqd10 and klcr2 had WT-like seeds. The cellulose defects of i9 i10 and klcr1 mutants were rescued by IQD9 and KLCR1 transgene complementation, respectively, using their native promoters (Fig. 3a). While all the iqd9, klcr1 and trm4 mutant combinations examined showed short S4B-stained cellulosic rays compared to WT (Fig. 3 and Fig. S3), counterstaining of mucilage with calcofluor displayed relatively normal content of other β-glucans (Fig. S3). Despite evident changes in the architecture of cellulose extruded from hydrated seeds, these MT-related mutants did not alter the crystalline cellulose content of whole seeds (Fig. 3c). Mutations in IQD9 or KLCR1 did not alter seed shape (Fig. S4), nor the germination rate or sensitivity to salt (Fig. S5).

Fig. 3
Fig. 3

IQD9 and KLCR1 are important for cellulose deposition around seed surface. (a) S4B-stained cellulosic rays in mucilage capsules. Bars = 100 µm. (b) The length of cellulosic rays stained with S4B. Boxes show the 25–75% quartiles, the median value (inner horizontal line), and whiskers extending to the largest/smallest values (≥10 measurements per biological replicate). (c) Crystalline cellulose content in whole seeds (5 biological replicates per genotype, except 3 for muci10). Different letters in (b) and (c) mark significant changes (one-way ANOVA with Tukey test, P < 0.01).

Therefore, like TRM4 (Yang et al., 2019), the expression of IQD9 and KLCR1 in seeds primarily affect mucilage polymer organization.

IQD9 and IQD10 are also expressed beyond the seed coat

Based on promoter:GUS reporter fusions, pIQD9 and pIQD10 promoters were active in multiple plant tissues, including the vasculature system (shoot and roots), developing flowers and siliques (Fig. S1). Except for anthers, pIQD9 showed higher activity than pIQD10 in reproductive organs and seeds (Fig. S2). IQD10 was expressed highest in Arabidopsis stems and its ortholog in Populus deltoides (PdIQD10) affects the development of the woody stem (Badmi et al., 2018). However, stem cross-sections of the iqd10 single mutants and the i9 i10 double mutant did not show the irregular xylem (irx) phenotype observed in secondary cell wall mutants such as irx14 (Fig. S6a). Furthermore, iqd stems had normal monosaccharide composition (Fig. S6b), while irx14 stems were xylan-deficient as previously described (Brown et al., 2007). IQD9 and IQD10 are thus not indispensable for the formation of xylem cells with thick secondary cell walls, or their functions could be masked by other IQDs.

IQD9 proteins associate with MTs and interact with KLCR1

To determine the subcellular localization of IQD9 proteins, we first co-expressed IQD9-GFP fusion proteins and the MT marker RFP-TUB6 in N. benthamiana leaf epidermal cells. IQD9-GFP localized in striated arrays that overlapped with RFP-TUB6 at the cell cortex (Fig. S7), and could be abolished by treating cells with MT-depolymerizing oryzalin. Next, we co-expressed IQD9-GFP with mCherry-tagged KLCR1 (mCherry-KLCR1) in N. benthamiana and found that they were co-localized in arrays resembling MTs (Fig. 4a–c). IQD9 and KLCR1 still co-localized when their striated patterns were disassembled by oryzalin treatment (Fig. 4c–e). We validated that these two proteins physically interact using co-immunoprecipitation (co-IP). IQD9-GFP and RFP-tagged KLCR1 (RFP-KLCR1) proteins were expressed in N. benthamiana leaves, extracted and purified by GFP-trap beads. Western blotting showed that all recombinant proteins were present in the input fractions (Fig. 4f), before the addition of the GFP-binding beads. RFP-KLCR1 proteins were detected on GFP-Trap beads only when co-expressed with IQD9-GFP, but not with GFP alone. Therefore, IQD9 proteins physically interacted with KLCR1 in vivo and were closely associated with cortical MTs.

Fig. 4
Fig. 4

IQD9 co-aligns and interacts with KLCR1. (a) IQD9-GFP localized in cortical arrays in N. benthamiana cells. (b) RFP-KLCR1 shows diffuse localization when overexpressed on its own in tobacco. (c) Subcellular co-localization of IQD9-GFP and mCherry-KLCR1 in the mock and oryzalin-treated tobacco epidermal cells. Both transiently expressed proteins were oryzalin-sensitive. (d) Fluorescent intensity plot along the dashed line in (c). Bars = 10 µm. (e) Pearson correlation coefficient between IQD9-GFP and mCherry-KLCR1 in (c), n=5 cells from 5 independent treatments. (f) Co-IP of proteins transiently expressed in tobacco leaves. Colored triangles marked the expected size of each protein. Labels: Input (protein supernatant before adding the GFP-Trap beads); Super (unbound supernatant after bead incubation); Wash (Supernatant from last wash step); Beads (co-IP proteins that tightly bind GFP-Trap).

Localization of IQD9-GFP during mucilage biosynthesis

To investigate the distribution of IQD9-GFP in Arabidopsis, we examined its subcellular localization under the control of its native promoter in the complemented iqd9 line, which rescued the mucilage defects (Fig. 1). While undetectable in young seedlings, IQD9-GFP fluorescence was evident during seed coat development (Fig. S8a), particularly at the peak stage of mucilage biosynthesis. Z-stack maximum projections displayed IQD9-GFP proteins in MT arrays, near the PM and inside the nucleus (Fig. 5a). IQD9-GFP displayed circular arrays around the cytoplasmic column, resembling previously described CESA trajectories during mucilage production (Griffiths et al., 2015). At SCE cell boundaries, IQD9-GFP proteins co-localized with the PM stained by FM4-64. Time-lapse imaging revealed that IQD9-GFP proteins were static (the vertical lines in kymograph; Fig. 5b), as previously noted for KLCR/CMU proteins (Liu et al., 2016). Highly immobile KLCR1-GFP proteins, expressed under its native promoter in the complemented klcr1 line, were also associated with both MTs and PM throughout SCE development (Fig. S8b), but lacked the nuclear localization observed for IQD9-GFP. In cross-sectional views of live SCE cells, both IQD9-GFP and KLCR1-GFP were localized primarily as striated arrays adjacent to the mucilage pocket (Fig. 5c,d).

Fig. 5
Fig. 5

IQD9 and KLCR1 localization during mucilage biosynthesis in complemented lines. (a) Z-stack maximum projection of IQD9-GFP SCE cells stained with FM4-64 at 7 DPA. IQD9 is localized at the PM, MTs and in a nuclear body (arrow). (b) Time-lapse of IQD9-GFP and kymograph along the dashed line. (c) Cross-sectional views of SCE cells expressing IQD9-GFP. The arrow marks a nuclear compartment. (d) Cross-sectional views of SCE cells expressing KLCR1-GFP during mucilage biosynthesis. Bars = 10 µm.

IQD9 maintains MT organization in SCE cells

Proper MT organization is essential for the establishment of mucilage architecture. The MT marker RFP-TUB6, which formed circular arrays around the cytoplasmic column of SCE cells at 7 DPA (Yang et al., 2019), was introduced into the iqd9-1 mutant by crossing. In contrast to the WT background, circular MT arrays were undetectable in all iqd9 SCE cells expressing RFP-TUB6 (Fig. 6). While MT organization was severely disrupted in the seed coat, both WT and iqd9 displayed transversely oriented RFP-TUB6 arrays in hypocotyl epidermal cells (Fig. S9), despite some variation in fluorescence intensity. Therefore, the distribution of cortical MTs in the seed coat depends heavily on IQD9, while cytoskeleton organization in other tissues likely requires additional IQDs.

Fig. 6
Fig. 6

MT organization was impaired in iqd9 SCE cells. Z-stack maximum projections of RFP-TUB6 in the SCE cells at 7 DPA stage. RFP-TUB6 cortical arrays formed in the WT background but not in iqd9 SCE cells. WT cells without RFP-TUB6 served as a negative control. Bars = 50 µm.

The loss of IQD9 reduces CESA3 velocity

CESA3 is a key subunit of the CSC that polarly deposits cellulose in seed mucilage pockets (Griffiths et al., 2015). Since cellulose distribution is disordered in iqd9 mucilage (Fig. 3), we hypothesized that IQD9 influences CSC motility at the cell cortex. Consistent with previous results (Griffiths et al., 2015), time-lapse images revealed GFP-CESA3 proteins moved in a unidirectional, clockwise manner around the cytoplasmic column of SCE cells (Fig. 7). While this pattern was still present, the velocity of GFP-CESA3 particles decreased from 135.9 ± 8.2 nm min-1 in WT cells to only 92.8 ± 17.3 nm min-1 in iqd9 (mean ± SD; at least 170 measurements of 9 cells from 3 plants per genotype). Consistent with their mucilage staining phenotypes (Fig. 1, Fig. S2 and Fig. S3), the movement of GFP-CESA3 was also reduced in trm4 cells, akin to iqd9 (Fig. 7). In these mutant seeds, CSC movement appeared to be uncoupled from MTs, a behavior previously described for CESA proteins in klcr (cmu) mutants (Liu et al., 2016), but could not be monitored in greater detail due to the severe disruption of RFP-TUB6 localization in SCE cells (Fig. 6; Yang et al., 2019). Taken together, IQD9 is a novel protein required for seed mucilage biosynthesis by maintaining cortical MT arrays and the speed of CESA movement, which influence cellulose distribution.

Fig. 7
Fig. 7

IQD9 and TRM4 enhance the velocity of GFP-CESA3 proteins.

(a) Single and time-lapse images (acquired every 5 s for 300 s) of GFP-CESA3 in WT, iqd9 and trm4 SCE cells at 7 DPA. The bottom row shows kymographs of GFP-CESA3 from dashed lines in the middle row. Bars = 10 µm. (b) Mean GFP-CESA3 velocity in 9 cells from three plants per genotype. Letters label significant differences (one-way ANOVA with Tukey test, P< 0.01). (c) Distribution of GFP-CESA3 velocities for analyzed particles (N=209 for WT, 170 for iqd9 and 131 for trm4).

Discussion

In the past decade, SCE cells have become a popular model to identify and study cell wall regulators as well as carbohydrate-active enzymes. Dozens of mucilage-related genes have been gradually characterized in Arabidopsis (Voiniciuc et al., 2015c; Šola et al., 2019), primarily through forward and reverse genetic screens. In addition to mutants generated in the laboratory, the architecture of mucilage β-glucans was found to vary dramatically in natural populations of Arabidopsis thaliana (Sullivan et al., 2011; North et al., 2014; Voiniciuc et al., 2016). The Arabidopsis research findings have been accompanied by advances in the mucilage structure of food crops such as Linum usitatissimum (flax; Viudes et al., 2020) and Plantago ovata (psyllium; (Cowley & Burton, 2021), which contain a higher proportion of non-pectic polymers. Despite the evolution of various mucilage traits within the Brassicaceae family (Viudes et al., 2021), how MTs regulate the intricate organization of this specialized secondary cell wall has remained a relatively blank slate. The Arabidopsis genome encodes hundreds of putative MT-associated proteins, but only MOR1 and TRM4 were previously shown to influence seed mucilage synthesis (McFarlane et al., 2008; Hamada, 2014; Yang et al., 2019). Additional MT-associated proteins (e.g. CSI1/POM2; CC1; IQD13 and KLCRs/CMUs) involved in cell wall biosynthesis were characterized in other tissues (Li et al., 2012; Bringmann et al., 2012b; Endler et al., 2015; Liu et al., 2016; Sugiyama et al., 2017), so the players that guide mucilage biosynthesis remained unclear.

IQD9 sustains MT organization during specialized cell wall deposition

In this study, we discovered that IQD9 and its interactor KLCR1 localize to cortical arrays that resemble the circular paths of MTs (Fig. 6) and multiple CESAs (Griffiths et al., 2015) during mucilage biosynthesis. IQD9 co-localized with MTs and was sensitive to their depolymerization by oryzalin, suggesting that IQD9 may be capable of directly binding MTs like other family members. The DUF4005 domain of IQD16 was recently shown to mediate MT binding in vivo as well as in vitro (Li et al., 2021). As one of the shortest family members (Abel et al., 2005), IQD9 lacks the DUF4005 domain but contains a region similar to the MT2 domain of IQD13 (Fig. S1c), which is sufficient for MT localization in vivo (Sugiyama et al., 2017). The highly immobile IQD9 proteins could function similarly to KLCR1/CMU1, its binding partner (Fig. 4), to stabilize the cortical MT arrays of SCE cells and sustain CSC speed during cellulose deposition. Consistent with this hypothesis, oryzalin treatment of SCE cells severely disrupted the trajectory and velocity of GFP-CESA3 (Griffiths et al., 2015). The reduced velocity of CESA3-containing CSCs in iqd9 and trm4 SCE cells (Fig. 7) shows that multiple classes of proteins are required to shape the circular MT arrays and cellulose distribution.

MT organization primarily affects cellulose distribution in seed mucilage

In Arabidopsis, SCE cells display MT and CSC dynamics that are considerably different from those of hypocotyl and protoxylem vessels (Griffiths et al., 2015; Watanabe et al., 2015; Griffiths & North, 2017), which serve as primary and secondary cell wall models. Both hypocotyl or protoxylem cells show transverse MTs arrays or bundles, aligned with bidirectional movement of CSCs in the PM. The velocities of CSCs during cellulose deposition range from 200–300 nm min-1 in the hypocotyl to 300–400 nm min-1 in the protoxylem. By contrast, SCE cells display circular MT arrays around the columella, aligned with unidirectional movement of CSCs with a velocity of 80 to 120 nm min-1 (Fig. 7, Griffiths et al., 2015). These unique MT pattern and CSC movements could lead to the polarized deposition of unusual cellulosic coils, which unwind with the expansion of pectin polymers during Arabidopsis seed hydration (Šola et al., 2019). Cytoskeletal defects in iqd9, klcr1, or trm4 mutants could slow CSC movement to result in shorter cellulose microfibrils that cannot extend to form long ray-like structures during mucilage release. The compact mucilage phenotypes of MT-related mutants and galactoglucomannan-deficient seeds such as muci10 could be explained by similar deficiencies in the assembly of cellulose chains (Yu et al., 2014; Voiniciuc et al., 2015b; Griffiths & North, 2017; Yang et al., 2019), Since relatively abundant MTs line the PM of the mucilage pocket where mucilage secretion occurs (McFarlane et al., 2008), MTs could also potentially target the secretion of pectin and hemicelluloses to the apoplast. However, a temperature-sensitive mutation of MOR1 partially disrupted mucilage release without clearly affecting the secretion of vesicles to the mucilage pocket and mucilage polymer accumulation (McFarlane et al., 2008). Despite severely disrupted RFP-TUB6 localization in SCE cells (Fig. 6; Yang et al., 2019), the iqd9 and trm4 seeds released matrix polysaccharides with a composition that was similar to WT (Fig. 2). Even though the iqd10-1 mutation partially reduced certain monosaccharides (Fig. 2), the mucilage released from these seeds showed a WT-like appearance (Figs. 1 and 3). Therefore, cortical MTs appear to have a relatively minor impact on the incorporation of pectin and hemicelluloses into the mucilage pockets of SCE cells. Unaltered mucilage adherence to the surface of iqd9, klcr1 and trm4 seeds is likely mediated by the presence of xylan (Voiniciuc et al., 2015a; Ralet et al., 2016), the SOS5 arabinogalactan protein and the receptor-like kinase FEI2 (Harpaz-Saad et al., 2011; Griffiths et al., 2016; Šola et al., 2019).

IQD, KLCR, and TRM proteins have interconnected functions

Our results and recently published findings indicate that distinct IQD proteins function in a tissue-specific manner by interacting with KLCR proteins and have potentially overlapping roles with TRM scaffolding proteins. Based on double mutant analyses (Figs. S2 and S3), the roles of IQD9 in SCE cells are nearly identical to those of KLCR1 and TRM4. Therefore, the encoded proteins could be associated as part of a single complex or pathway at the cell cortex. Some Arabidopsis IQDs (GFP-IQD1 or GFP-IQD2) can recruit KLCR1 to MTs when transiently co-expressed in tobacco cells (Bürstenbinder et al., 2013; Zang et al., 2021). The MT recruitment is consistent with our results for the transient expression of IQD9-GFP and KLCR1 (which behaves similarly when tagged with either RFP or mCherry), even though KLCR1/CMU1 alone can at least partially bind MTs (Liu et al., 2016; Zang et al., 2021). Furthermore, the KLCR1-IQD2 pair was recently shown to interact with the actin binding protein NET3C to modulate the shape of the endoplasmic reticulum at PM contact sites (Zang et al., 2021). We hypothesize that IQD9, KLCR1, TRM4 could be an integral part of an expanding group of proteins (Polko & Kieber, 2019) that support PM-bound CSC movement along the orientation of MT tracks. IQDs are hypothesized to function as scaffolds and may be modified by TON1/TRM/PP2A (TTP)-mediated dephosphorylation (Kumari et al., 2021). Via IQ67-domain-mediated calmodulin binding (Abel et al., 2005), IQDs could also participate in Ca2+ signaling to ultimately influence plant cell wall dynamics.

Intriguing roles of IQDs during secondary cell wall formation

Even though IQD9 and IQD10 promoters were active in both vegetative and reproductive organs (Fig. S1), IQD9 was indispensable only for the organization of seed mucilage polysaccharides (Figs. 1–3). While the expression of IQD9-GFP under its native promoter was detected only in the general seed coat (Fig. 5 and Fig. S4), KLCR1-GFP was expressed more ubiquitously in complemented lines. The transcription of IQD9, IQD10 and IQD13 was previously associated with secondary cell wall biosynthesis (Mutwil et al., 2008). Even though we detected transcriptional activity in the vasculature (Fig. S1), the absence of IQD9 and/or IQD10 did not cause irx phenotypes found in stems with defective cellulose-hemicellulose networks (Fig. S6; Brown et al., 2007). Their vascular functions could be masked by the expression of related genes such as IQD13, which was already shown to modulate MT organization during xylem cell formation (Sugiyama et al., 2017). In poplar, the down-regulation of PdIQD10 during wood formation increased the tree height, diameter and relative cellulose content (Badmi et al., 2018). Since PdIQD10 interacted with PdKLCRs and could be directed to the nucleus, the elevated cellulosic biomass of the transgenic trees suggests that IQD-KLCRs participate in a tight feedback-loop that regulates cellulose biosynthesis (Badmi et al., 2018). In Arabidopsis, multiple IQDs likely have redundant roles during stem development so higher-order mutants would be needed to decipher how IQD-KLCR are involved in signaling pathways or in direct interactions with secondary wall CSCs.

Future avenues to tailor cellulose deposition

The localization of soluble IQD9 and KLCR1/CMU1 (Fig. 5, Liu et al., 2016) proteins near or at the PM suggests that they could interact with membrane-bound CSC components, which travel in a spiral pattern during mucilage synthesis (Griffiths et al., 2015). TRM4 was previously shown to maintain MT organization and directly bind CESA3 (Yang et al., 2019) to enhance its mobility (Fig. 7). Although the mechanism that connects IQD9 and KLCR1 to TRM4 requires further investigation, we provide the first evidence that members of these three MT-associated families cooperate to direct cellulose deposition. Additional CSC-related genes are expressed during mucilage production (Griffiths & North, 2017), but their putative roles in mucilage biosynthesis remain to be investigated. Exploring the interactome of IQD9, KLCR1, and TRM4 could reveal novel targets to fine-tune the biosynthesis of cellulose, the most abundant renewable material on our planet. In addition to plant studies, the growing arsenal of proteins found to influence cellulose biosynthesis could be rapidly expressed and engineered in surrogate hosts (Pauly et al., 2019). Yeast species such as Pichia pastoris have already been used to express a Populus CESA capable of producing cellulose microfibrils in vitro (Purushotham et al., 2016) and to identify essential protein co-factors for CESA-like enzymes that catalyze hemicellulose elongation (Voiniciuc et al., 2019). Therefore, synthetic biology advances combined with attractive plant models, such as the Arabidopsis SCE cells, provide exciting avenues to refine the fibers that shape plants and many industrial products.

Authors Contributions

C.V. and K.B. conceived the project. B.Y. performed most of the experiments under C.V.’s supervision. G.S. and K.B. contributed genetic materials and performed RT-PCR, GUS and co-IP assays. B.Y. and C.V. analyzed data and wrote the manuscript with input from all authors.

Acknowledgements

We thank Tilman Jacob, Romina Plötner and Kristina Rosenzweig for technical assistance and colleagues at the Leibniz Institute of Plant Biochemistry for microscope access. The pUBQ:RFP-TUB6, pKLCR1:KLCR1-GFP in klcr1-1 seeds and the proCESA3:GFP-CESA3 in je5 seeds were provided by Geoffrey Wasteneys (University of British Columbia, CA), Pengwei Wang (Huazhong Agricultural University, CN) and Samantha Vernhettes (Université Paris-Saclay, FR), respectively. This work was supported by core funding (Leibniz Association) from the Federal Republic of Germany and the state of Saxony-Anhalt, and by DFG grants (BU2955/2-1 and BU2955/1-1 to K.B.; 414353267 to C.V.).

Footnotes

  • E-Mails

    B.Y. Bo.Yang{at}ipb-halle.de

    G.S. Gina.Stamm{at}ipb-halle.de

    K.B. Katharina.Buerstenbinder{at}ipb-halle.de

    C.V. Catalin.Voiniciuc{at}ipb-halle.de

References

  1. ↵
    Abel S, Savchenko T, Levy M. 2005. Genome-wide comparative analysis of the IQD gene families in Arabidopsis thaliana and Oryza sativa. BMC Evolutionary Biology 5: 72.
    OpenUrl
  2. ↵
    Ambrose C, Allard JF, Cytrynbaum EN, Wasteneys GO. 2011. A CLASP-modulated cell edge barrier mechanism drives cell-wide cortical microtubule organization in Arabidopsis. Nature Communications 2: 430.
    OpenUrl
  3. ↵
    Anderson CT, Carroll A, Akhmetova L, Somerville C. 2010. Real-Time Imaging of Cellulose Reorientation during Cell Wall Expansion in Arabidopsis Roots. Plant Physiology 152: 787–796.
    OpenUrlAbstract/FREE Full Text
  4. ↵
    Badmi R, Payyavula RS, Bali G, Guo H-B, Jawdy SS, Gunter LE, Yang X, Winkeler KA, Collins C, Rottmann WH, et al. 2018. A New Calmodulin-Binding Protein Expresses in the Context of Secondary Cell Wall Biosynthesis and Impacts Biomass Properties in Populus. Frontiers in Plant Science 9.
  5. ↵
    Ben-Tov D, Abraham Y, Stav S, Thompson K, Loraine A, Elbaum R, de Souza A, Pauly M, Kieber JJ, Harpaz-Saad S. 2015. COBRA-LIKE2, a Member of the Glycosylphosphatidylinositol-Anchored COBRA-LIKE Family, Plays a Role in Cellulose Deposition in Arabidopsis Seed Coat Mucilage Secretory Cells,. Plant Physiology 167: 711–724.
    OpenUrlAbstract/FREE Full Text
  6. ↵
    Ben-Tov D, Idan-Molakandov A, Hugger A, Ben-Shlush I, Günl M, Yang B, Usadel B, Harpaz-Saad S. 2018. The role of COBRA-LIKE 2 function, as part of the complex network of interacting pathways regulating Arabidopsis seed mucilage polysaccharide matrix organization. The Plant Journal 94: 497–512.
    OpenUrl
  7. ↵
    Bringmann M, Landrein B, Schudoma C, Hamant O, Hauser M-T, Persson S. 2012a. Cracking the elusive alignment hypothesis: the microtubule–cellulose synthase nexus unraveled. Trends in Plant Science 17: 666–674.
    OpenUrlCrossRefPubMedWeb of Science
  8. ↵
    Bringmann M, Li E, Sampathkumar A, Kocabek T, Hauser M-T, Persson S. 2012b. POM-POM2/CELLULOSE SYNTHASE INTERACTING1 Is Essential for the Functional Association of Cellulose Synthase and Microtubules in Arabidopsis. The Plant Cell 24: 163–177.
    OpenUrlAbstract/FREE Full Text
  9. ↵
    Brown DM, Goubet F, Wong VW, Goodacre R, Stephens E, Dupree P, Turner SR. 2007. Comparison of five xylan synthesis mutants reveals new insight into the mechanisms of xylan synthesis. The Plant Journal 52: 1154–1168.
    OpenUrlCrossRefPubMedWeb of Science
  10. ↵
    Bürstenbinder K, Möller B, Plötner R, Stamm G, Hause G, Mitra D, Abel S. 2017. The IQD Family of Calmodulin-Binding Proteins Links Calcium Signaling to Microtubules, Membrane Subdomains, and the Nucleus. Plant Physiology 173: 1692–1708.
    OpenUrlAbstract/FREE Full Text
  11. ↵
    Bürstenbinder K, Savchenko T, Müller J, Adamson AW, Stamm G, Kwong R, Zipp BJ, Dinesh DC, Abel S. 2013. Arabidopsis Calmodulin-binding Protein IQ67-Domain 1 Localizes to Microtubules and Interacts with Kinesin Light Chain-related Protein-1. Journal of Biological Chemistry 288: 1871–1882.
    OpenUrlAbstract/FREE Full Text
  12. ↵
    Cowley JM, Burton RA. 2021. The goo-d stuff: Plantago as a myxospermous model with modern utility. New Phytologist 229: 1917–1923.
    OpenUrl
  13. ↵
    Crowell EF, Timpano H, Desprez T, Franssen-Verheijen T, Emons A-M, Höfte H, Vernhettes S. 2011. Differential Regulation of Cellulose Orientation at the Inner and Outer Face of Epidermal Cells in the Arabidopsis Hypocotyl. The Plant Cell 23: 2592–2605.
    OpenUrlAbstract/FREE Full Text
  14. ↵
    Desprez T, Juraniec M, Crowell EF, Jouy H, Pochylova Z, Parcy F, Hofte H, Gonneau M, Vernhettes S. 2007. Organization of cellulose synthase complexes involved in primary cell wall synthesis in Arabidopsis thaliana. Proceedings of the National Academy of Sciences 104: 15572–15577.
    OpenUrlAbstract/FREE Full Text
  15. ↵
    1. Anderson CT,
    2. Haswell ES,
    3. Dixit R
    Duncombe SG, Barnes WJ, Anderson CT. 2020. Chapter 11 - Imaging the delivery and behavior of cellulose synthases in Arabidopsis thaliana using confocal microscopy. In: Anderson CT, Haswell ES, Dixit R, eds. Plant Cell Biology. Methods in Cell Biology. Academic Press, 201–213.
  16. ↵
    Endler A, Kesten C, Schneider R, Zhang Y, Ivakov A, Froehlich A, Funke N, Persson S. 2015. A Mechanism for Sustained Cellulose Synthesis during Salt Stress. Cell 162: 1353–1364.
    OpenUrlCrossRefPubMed
  17. ↵
    Foster CE, Martin TM, Pauly M. 2010. Comprehensive Compositional Analysis of Plant Cell Walls (Lignocellulosic biomass) Part II: Carbohydrates. Journal of Visualized Experiments: 1837.
  18. ↵
    Ganguly A, Zhu C, Chen W, Dixit R. 2020. FRA1 Kinesin Modulates the Lateral Stability of Cortical Microtubules through Cellulose Synthase–Microtubule Uncoupling Proteins. The Plant Cell 32: 2508–2524.
    OpenUrlAbstract/FREE Full Text
  19. ↵
    Gantner J, Ordon J, Ilse T, Kretschmer C, Gruetzner R, Löfke C, Dagdas Y, Bürstenbinder K, Marillonnet S, Stuttmann J. 2018. Peripheral infrastructure vectors and an extended set of plant parts for the Modular Cloning system. PLOS ONE 13: e0197185.
    OpenUrl
  20. ↵
    Grefen C, Chen Z, Honsbein A, Donald N, Hills A, Blatt MR. 2010. A Novel Motif Essential for SNARE Interaction with the K+ Channel KC1 and Channel Gating in Arabidopsis. The Plant Cell 22: 3076–3092.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    Griffiths JS, Crepeau M-J, Ralet M-C, Seifert GJ, North HM. 2016. Dissecting Seed Mucilage Adherence Mediated by FEI2 and SOS5. Frontiers in Plant Science 7: 1–13.
    OpenUrl
  22. ↵
    Griffiths JS, North HM. 2017. Sticking to cellulose: exploiting Arabidopsis seed coat mucilage to understand cellulose biosynthesis and cell wall polysaccharide interactions. New Phytologist 214: 959–966.
    OpenUrlCrossRef
  23. ↵
    Griffiths JS, Šola K, Kushwaha R, Lam P, Tateno M, Young R, Voiniciuc C, Dean G, Mansfield SD, DeBolt S, et al. 2015. Unidirectional Movement of Cellulose Synthase Complexes in Arabidopsis Seed Coat Epidermal Cells Deposit Cellulose Involved in Mucilage Extrusion, Adherence, and Ray Formation. Plant Physiology 168: 502–520.
    OpenUrlAbstract/FREE Full Text
  24. ↵
    Griffiths JS, Tsai AYL, Xue H, Voiniciuc C, Šola K, Seifert GJ, Mansfield SD, Haughn GW. 2014. SALT-OVERLY SENSITIVE5 mediates arabidopsis seed coat mucilage adherence and organization through pectins. Plant Physiology 165: 991–1004.
    OpenUrlAbstract/FREE Full Text
  25. ↵
    Gutierrez R, Lindeboom JJ, Paredez AR, Emons AMC, Ehrhardt DW. 2009. Arabidopsis cortical microtubules position cellulose synthase delivery to the plasma membrane and interact with cellulose synthase trafficking compartments. Nature Cell Biology 11: 797–806.
    OpenUrlCrossRefPubMedWeb of Science
  26. ↵
    Hamada T. 2014. Microtubule Organization and Microtubule-Associated Proteins in Plant Cells. In: International Review of Cell and Molecular Biology. Elsevier, 1–52.
  27. ↵
    Harpaz-Saad S, McFarlane HE, Xu S, Divi UK, Forward B, Western TL, Kieber JJ. 2011. Cellulose synthesis via the FEI2 RLK/SOS5 pathway and CELLULOSE SYNTHASE 5 is required for the structure of seed coat mucilage in Arabidopsis: Synthesis of cellulose in seed mucilage. The Plant Journal 68: 941–953.
    OpenUrlCrossRefPubMedWeb of Science
  28. ↵
    Hu R, Li J, Wang X, Zhao X, Yang X, Tang Q, He G, Zhou G, Kong Y. 2016. Xylan synthesized by Irregular Xylem 14 (IRX14) maintains the structure of seed coat mucilage in Arabidopsis. Journal of Experimental Botany 67: 1243–1257.
    OpenUrlCrossRefPubMed
  29. ↵
    Karimi M, Inzé D, Depicker A. 2002. GATEWAY vectors for Agrobacterium-mediated plant transformation. Trends in Plant Science 7: 193–195.
    OpenUrlCrossRefPubMedWeb of Science
  30. ↵
    Kölling M, Kumari P, Bürstenbinder K. 2019. Calcium-and calmodulin-regulated microtubule-associated proteins as signal-integration hubs at the plasma membrane-cytoskeleton nexus. Journal of Experimental Botany 70: 387–396.
    OpenUrl
  31. ↵
    Kumari P, Dahiya P, Livanos P, Zergiebel L, Kölling M, Poeschl Y, Stamm G, Hermann A, Abel S, Müller S, et al. 2021. IQ67 DOMAIN proteins facilitate preprophase band formation and division-plane orientation. Nature Plants: 1–9.
  32. ↵
    Li Y, Huang Y, Wen Y, Wang D, Liu H, Li Y, Zhao J, An L, Yu F, Liu X. 2021. The domain of unknown function 4005 (DUF4005) in an Arabidopsis IQD protein functions in microtubule binding. Journal of Biological Chemistry 297: 100849.
    OpenUrl
  33. ↵
    Li S, Lei L, Somerville CR, Gu Y. 2012. Cellulose synthase interactive protein 1 (CSI1) links microtubules and cellulose synthase complexes. Proceedings of the National Academy of Sciences 109: 185–190.
    OpenUrlAbstract/FREE Full Text
  34. ↵
    Liang H, Zhang Y, Martinez P, Rasmussen CG, Xu T, Yang Z. 2018. The Microtubule-Associated Protein IQ67 DOMAIN5 Modulates Microtubule Dynamics and Pavement Cell Shape. Plant Physiology 177: 1555–1568.
    OpenUrlAbstract/FREE Full Text
  35. ↵
    Liu Z, Schneider R, Kesten C, Zhang Y, Somssich M, Zhang Y, Fernie AR, Persson S. 2016. Cellulose-Microtubule Uncoupling Proteins Prevent Lateral Displacement of Microtubules during Cellulose Synthesis in Arabidopsis. Developmental Cell 38: 305–315.
    OpenUrlCrossRef
  36. ↵
    Lloyd C, Hussey P. 2001. Microtubule-associated proteins in plants — why we need a map. Nature Reviews Molecular Cell Biology 2: 40–47.
    OpenUrlCrossRefPubMedWeb of Science
  37. ↵
    McFarlane HE, Young RE, Wasteneys GO, Samuels AL. 2008. Cortical microtubules mark the mucilage secretion domain of the plasma membrane in Arabidopsis seed coat cells. Planta 227: 1363–1375.
    OpenUrlCrossRefPubMedWeb of Science
  38. ↵
    Mendu V, Griffiths JS, Persson S, Stork J, Downie AB, Voiniciuc C, Haughn GW, DeBolt S. 2011. Subfunctionalization of Cellulose Synthases in Seed Coat Epidermal Cells Mediates Secondary Radial Wall Synthesis and Mucilage Attachment. Plant Physiology 157: 441–453.
    OpenUrlAbstract/FREE Full Text
  39. ↵
    Mielke S, Zimmer M, Meena MK, Dreos R, Stellmach H, Hause B, Voiniciuc C, Gasperini D. 2021. Jasmonate biosynthesis arising from altered cell walls is prompted by turgor-driven mechanical compression. Science Advances 7: eabf0356.
    OpenUrlFREE Full Text
  40. ↵
    Mitra D, Klemm S, Kumari P, Quegwer J, Möller B, Poeschl Y, Pflug P, Stamm G, Abel S, Bürstenbinder K. 2019. Microtubule-associated protein IQ67 DOMAIN5 regulates morphogenesis of leaf pavement cells in Arabidopsis thaliana. Journal of Experimental Botany 70: 529–543.
    OpenUrl
  41. ↵
    Mutwil M, Obro J, Willats WGT, Persson S. 2008. GeneCAT--novel webtools that combine BLAST and co-expression analyses. Nucleic Acids Research 36: W320–W326.
    OpenUrlCrossRefPubMedWeb of Science
  42. ↵
    Nakagawa T, Suzuki T, Murata S, Nakamura S, Hino T, Maeo K, Tabata R, Kawai T, Tanaka K, Niwa Y, et al. 2007. Improved Gateway binary vectors: high-performance vectors for creation of fusion constructs in transgenic analysis of plants. Bioscience, Biotechnology, and Biochemistry 71: 2095–2100.
    OpenUrlCrossRefPubMedWeb of Science
  43. ↵
    North HM, Berger A, Saez-Aguayo S, Ralet M-C. 2014. Understanding polysaccharide production and properties using seed coat mutants: future perspectives for the exploitation of natural variants. Annals of Botany 114: 1251–1263.
    OpenUrlCrossRefPubMed
  44. ↵
    Paredez AR, Somerville CR, Ehrhardt DW. 2006. Visualization of Cellulose Synthase Demonstrates Functional Association with Microtubules. Science 312: 1491–1495.
    OpenUrlAbstract/FREE Full Text
  45. ↵
    Pauly M, Gawenda N, Wagner C, Fischbach P, Ramírez V, Axmann IM, Voiniciuc C. 2019. The Suitability of Orthogonal Hosts to Study Plant Cell Wall Biosynthesis. Plants 8: 516.
    OpenUrl
  46. ↵
    Polko JK, Barnes WJ, Voiniciuc C, Doctor S, Steinwand B, Hill JL, Tien M, Pauly M, Anderson CT, Kieber JJ. 2018. SHOU4 Proteins Regulate Trafficking of Cellulose Synthase Complexes to the Plasma Membrane. Current Biology 28: 3174–3182.e6.
    OpenUrlCrossRefPubMed
  47. ↵
    Polko JK, Kieber JJ. 2019. The Regulation of Cellulose Biosynthesis in Plants. The Plant Cell 31: 282–296.
    OpenUrlAbstract/FREE Full Text
  48. ↵
    Purushotham P, Cho SH, Díaz-Moreno SM, Kumar M, Nixon BT, Bulone V, Zimmer J. 2016. A single heterologously expressed plant cellulose synthase isoform is sufficient for cellulose microfibril formation in vitro. Proceedings of the National Academy of Sciences 113: 11360–11365.
    OpenUrlAbstract/FREE Full Text
  49. ↵
    Ralet M-C, Crépeau M-J, Vigouroux J, Tran J, Berger A, Sallé C, Granier F, Botran L, North HM. 2016. Xylans Provide the Structural Driving Force for Mucilage Adhesion to the Arabidopsis Seed Coat. Plant Physiology 171: 165–178.
    OpenUrlAbstract/FREE Full Text
  50. ↵
    Sedbrook JC, Kaloriti D. 2008. Microtubules, MAPs and plant directional cell expansion. Trends in Plant Science 13: 303–310.
    OpenUrlCrossRefPubMedWeb of Science
  51. ↵
    Šola K, Dean GH, Haughn GW. 2019. Arabidopsis Seed Mucilage: A Specialised Extracellular Matrix that Demonstrates the Structure–Function Versatility of Cell Wall Polysaccharides. Annual Plant Reviews online 2: 1085–1116.
    OpenUrl
  52. ↵
    Sugiyama Y, Wakazaki M, Toyooka K, Fukuda H, Oda Y. 2017. A Novel Plasma Membrane-Anchored Protein Regulates Xylem Cell-Wall Deposition through Microtubule-Dependent Lateral Inhibition of Rho GTPase Domains. Current Biology 27: 2522–2528.e4.
    OpenUrlCrossRef
  53. ↵
    Sullivan S, Ralet M-C, Berger A, Diatloff E, Bischoff V, Gonneau M, Marion-Poll A, North HM. 2011. CESA5 Is Required for the Synthesis of Cellulose with a Role in Structuring the Adherent Mucilage of Arabidopsis Seeds. Plant Physiology 156: 1725–1739.
    OpenUrlAbstract/FREE Full Text
  54. ↵
    Updegraff DM. 1969. Semimicro determination of cellulose inbiological materials. Analytical Biochemistry 32: 420–424.
    OpenUrlCrossRefPubMedWeb of Science
  55. ↵
    Vellosillo T, Yeats T, Sorek N. 2015. Analysis of in vivo Cellulose Biosynthesis in Arabidopsis Cells by Spinning Disk Confocal Microscopy. Bio-protocol 5: e1617–e1617.
    OpenUrl
  56. ↵
    Viudes S, Burlat V, Dunand C. 2020. Seed mucilage evolution: Diverse molecular mechanisms generate versatile ecological functions for particular environments. Plant, Cell & Environment 43: 2857–2870.
    OpenUrlCrossRef
  57. ↵
    Viudes S, Dunand C, Burlat V. 2021. Myxospermy Evolution in Brassicaceae: A Highly Complex and Diverse Trait with Arabidopsis as an Uncommon Model. Cells 10: 2470.
    OpenUrl
  58. ↵
    Voiniciuc C. 2016. Quantification of the Mucilage Detachment from Arabidopsis Seeds. BIO-PROTOCOL 6: 1–9.
    OpenUrl
  59. ↵
    Voiniciuc C, Dama M, Gawenda N, Stritt F, Pauly M. 2019. Mechanistic insights from plant heteromannan synthesis in yeast. Proceedings of the National Academy of Sciences 116: 522–527.
    OpenUrlAbstract/FREE Full Text
  60. ↵
    Voiniciuc C, Guenl M, Schmidt MH-W, Usadel B. 2015a. Highly Branched Xylan Made by IRX14 and MUCI21 Links Mucilage to Arabidopsis Seeds. Plant Physiology: pp.01441.2015.
  61. ↵
    Voiniciuc C, Günl M. 2016. Analysis of Monosaccharides in Total Mucilage Extractable from Arabidopsis Seeds. Bio-protocol 6: e1801–e1801.
    OpenUrl
  62. ↵
    Voiniciuc C, Schmidt MH-W, Berger A, Yang B, Ebert B, Scheller HV, North HM, Usadel B, Günl M. 2015b. MUCILAGE-RELATED10 Produces Galactoglucomannan That Maintains Pectin and Cellulose Architecture in Arabidopsis Seed Mucilage. Plant Physiology 169: 403–420.
    OpenUrlAbstract/FREE Full Text
  63. ↵
    Voiniciuc C, Yang B, Schmidt MH-W, Günl M, Usadel B. 2015c. Starting to Gel: How Arabidopsis Seed Coat Epidermal Cells Produce Specialized Secondary Cell Walls. International Journal of Molecular Sciences 16: 3452–3473.
    OpenUrl
  64. ↵
    Voiniciuc C, Zimmermann E, Schmidt MH-W, Günl M, Fu L, North HM, Usadel B. 2016. Extensive Natural Variation in Arabidopsis Seed Mucilage Structure. Frontiers in Plant Science 7: 1–14.
    OpenUrl
  65. ↵
    Watanabe Y, Meents MJ, McDonnell LM, Barkwill S, Sampathkumar A, Cartwright HN, Demura T, Ehrhardt DW, Samuels AL, Mansfield SD. 2015. Visualization of cellulose synthases in Arabidopsis secondary cell walls. Science 350: 198–203.
    OpenUrlAbstract/FREE Full Text
  66. ↵
    Yang B, Hofmann F, Usadel B, Voiniciuc C. 2021. Seed hemicelluloses tailor mucilage properties and salt tolerance. New Phytologist 229: 1946–1954.
    OpenUrl
  67. ↵
    Yang B, Voiniciuc C, Fu L, Dieluweit S, Klose H, Usadel B. 2019. TRM4 is essential for cellulose deposition in Arabidopsis seed mucilage by maintaining cortical microtubule organization and interacting with CESA3. New Phytologist 221: 881–895.
    OpenUrl
  68. ↵
    Yu L, Shi D, Li J, Kong Y, Yu Y, Chai G, Hu R, Wang J, Hahn MG, Zhou G. 2014. CELLULOSE SYNTHASE-LIKE A2, a glucomannan synthase, is involved in maintaining adherent mucilage structure in arabidopsis seed. Plant Physiology 164: 1842–1856.
    OpenUrlAbstract/FREE Full Text
  69. ↵
    Zang J, Klemm S, Pain C, Duckney P, Bao Z, Stamm G, Kriechbaumer V, Bürstenbinder K, Hussey PJ, Wang P. 2021. A novel plant actin-microtubule bridging complex regulates cytoskeletal and ER structure at ER-PM contact sites. Current Biology 31: 1251–1260.e4.
    OpenUrl
  70. ↵
    Zhang Y, Nikolovski N, Sorieul M, Vellosillo T, McFarlane HE, Dupree R, Kesten C, Schneider R, Driemeier C, Lathe R, et al. 2016. Golgi-localized STELLO proteins regulate the assembly and trafficking of cellulose synthase complexes in Arabidopsis. Nature Communications 7: 11656.
    OpenUrl
Back to top
Posted December 12, 2021.
Download PDF

Supplementary Material

Email
Microtubule-associated IQD9 guides cellulose synthase velocity to shape seed mucilage
Bo Yang, Gina Stamm, Katharina Bürstenbinder, Cătălin Voiniciuc
bioRxiv 2021.12.11.472226; doi: https://doi.org/10.1101/2021.12.11.472226
Reddit logo Twitter logo Facebook logo LinkedIn logo Mendeley logo
Citation Tools

Subject Area